Bemisia tabaci (Gennadius) MEAM1 (Hemiptera: Aleyrodidae) and Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), two important invasive species, are serious agricultural pests. In this study, a one-step, single tube, duplex polymerase chain reaction (PCR) procedure was developed to allow rapid, specific, and sensitive identification of B. tabaci MEAM1 and F. occidentalis in predator guts. The system and conditions used for the duplex PCR were optimized. The species specificity of the duplex PCR determined by comparison against non-targets that might interact with B. tabaci MEAM1 and F. occidentalis showed that oligonucleotide primers amplified nuclear gene target sequences present only in B. tabaci MEAM1 or F. occidentalis. The limits of detection were 9.53 ng μl−1 for B. tabaci MEAM1 and 8.94 ng μl−1 for F. occidentalis. Within a field cage study, in which predators Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) and Orius sauteri (Poppius) (Hemiptera: Anthocoridae) were allowed to feed on B. tabaci MEAM1 and F. occidentalis for 10 h, the B. tabaci MEAM1 DNA was detectable in 100% of H. axyridis and O. sauteri, and F. occidentalis DNA was detectable in 80% of H. axyridis and 90% of O. sauteri; this implicated that B. tabaci MEAM1 and F. occidentalis remains could be detected in native predator guts simultaneously. The accuracy and reliability of the assay suggested strongly that the duplex PCR optimized for B. tabaci MEAM1 and F. occidentalis is sensitive and specific for both invasive insects and is therefore useful in early diagnosis and monitoring of B. tabaci MEAM1 and F. occidentalis infections, and can be used to identify domestic predator species and food web relationships.